Method for treating neurodegenerative diseases

ABSTRACT

The present invention is directed to a method for treating a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS), Alzheimer disease, Parkinson&#39;s disease, Huntington&#39;s disease, frontotemporal degeneration, dementia with Lewy bodies, a motor neuron disease, or a demyelinating disease. The method comprises administering to a subject in need thereof a Ppargc1a activator 2-(4-tert-butylphenyl)-1H-benzimidazole, 2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, in an effective amount. A preferred route of administration is oral administration.

FIELD OF THE INVENTION

The present invention relates to methods for treating neurodegenerativediseases by administering to a subject a Ppargc1a activator,2-(4-tert-butylphenyl)-1H-benzimidazole,2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole.

BACKGROUND Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerativedisease that is characterized by the loss of motor neurons, leading toprogressive decline in motor function and ultimately death. The motorsymptoms of ALS include muscle weakness, twitching and wasting, whichleads to difficulties in speaking, swallowing and breathing. The causeof motor neuron death in ALS is unknown and 5-10% of the ALS cases areinherited.

Activation of immune cells in the central as well as peripheral nervoussystem has been suggested to be a critical determinant of diseaseprogression in ALS (Phani et al, Front Pharmacol. 3:150, 2012).Specifically, microglia and macrophages have been shown to play distinctroles in the orchestration of neuroinflammation in this disease (Dibajet al, PLoS One. 6(3):e17910, 2011; Boillee et al, Science, 312:1389-92,2006). Of note, bone marrow transplantation (BMT) to replace hostmyeloid cells has been shown to extend survival in an animal model ofALS, which was thought to be mediated by replacement of CNS microglia(Beers et al, Proc Natl Acad Sci USA. 103:16021-6, 2006). However,recent studies have shown that these cells do not develop from bonemarrow cells but from more primitive yolk sac progenitors (Ginhoux etal, Science, 330:841-5, 2110), suggesting that the bone marrow derivedcells that mediated the therapeutic effects of BMT in the study aboveare more likely peripheral or brain perivascular macrophages.Nevertheless, specific signaling pathways that contribute toinnate-immune-cell-mediated inflammation in ALS remain incompletelyunderstood.

Currently, there is no cure for ALS. Certain therapies such as riluzole,bone marrow transplantation (Deda, Cytotherapy. 11:18-25, 2009), andnon-invasive ventilation (McDermott et al, BMJ, 336:658-62, 2008) haveshown modest effects in improving quality of life and extendingsurvival, but none are curative or provide dramatic benefit.

Alzheimer's Disease

Alzheimer's Disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of motor function, in addition to memory,cognition, reasoning, judgment and emotional stability that graduallyleads to profound mental deterioration and ultimately death. Neuronalmetabolic dysfunction in the form of oxidative stress has been proposedto be an underlying cause of neurodegeneration in AD (Friedland-Leuneret al Mol Biol Transl Sci, 127:183-201, 2014).

Although AD develops differently for every individual, there are manycommon symptoms. Early symptoms are often mistakenly thought to beage-related concerns, or manifestations of stress. In the early stages,the most common symptoms are motor decline and difficulty in rememberingrecent events, known as short-term memory loss (Buchman et al, Exp RevNeurother, 11:665-76, 2011). When AD is suspected, the diagnosis isusually based on tests that evaluate behavior and thinking abilities,often followed by a brain scan if available. However, examination ofbrain tissue is required for a definitive diagnosis. As the diseaseadvances, symptoms can include confusion, irritability, aggression, moodswings, trouble with language, and long-term memory loss. As theperson's condition declines, he/she often withdraws from family andsociety. Gradually, bodily functions are lost, ultimately leading todeath.

Parkinson's Disease

Parkinson's disease (PD), also known as idiopathic or primaryparkinsonism, is a degenerative neurological disorder of the centralnervous system. The motor symptoms of PD result from the death ofdopamine-generating cells in the substantia nigra, a region of themidbrain; the cause of this cell death is unknown. Early in the courseof the disease, the most obvious symptoms are movement-related; theseinclude shaking, rigidity, slowness of movement and difficulty with finemotor skills, walking, and gait. Later, thinking and behavioral problemsmay arise, with dementia commonly occurring in the advanced stages ofthe disease, whereas depression is the most common psychiatric symptom.Other symptoms include sensory, sleep and emotional problems.

PD is characterized by progressive motor impairment andneuroinflammation induced by microglia, the resident immune cells of thecentral nervous system (Aguzzi et al, Science, 339:156-61, 2013).Inflammatory mediators produced by dysfunctional microglia have beenshown to induce neuronal cell death, which underlies the progressiveimpairment in cognitive and behavioral performance in neurodegenerativediseases (Czirr et al J Clin Invest, 122:1156-63, 2012). Nevertheless,specific signaling pathways that contribute to microglia-mediatedinflammation remain elusive.

Huntington's Disease

Huntington's disease (HD) is an autosomal dominant degenerative disorderof the central nervous system, in which the gene Huntington is mutated.HD is an inherited disease that causes the progressive breakdown(degeneration) of nerve cells in the brain. HD has a broad impact on aperson's functional abilities and usually results in movement, thinking(cognitive) and psychiatric disorders.

The symptoms of HD vary among affected subjects; however, theprogression of the disease is relatively predictable (Mason S et al, JNeurol. 2015). Early in the course of the disease, the symptoms aresubtle such as changes in mood. Later, cognition and motor problems mayarise, with dementia commonly occurring in the advanced stages of thedisease. Chorea (involuntary movement) is the most common motor symptom.Other complications include pneumonia, heart disease, and physicalinjuries due to falls.

There is currently no cure for HD and full time care is required forsubjects with advanced disease.

Frontotemporal Degeneration

Frontotemporal degeneration (FTD) is a disease that is closely relatedto AD in which progressive degeneration occurs in the frontal andtemporal lobes of the brain. Gliosis and inflammatory activation ofmicroglia have been documented in humans and animal models of FTD(Cagnin et al Annals of Neurol. 2004 6: 894-897; Yi et al. J. Exp. Med.2010. 1:117-128). Patients with FTD experience a gradual decline inbehavior and language with memory usually relatively preserved. As thedisease progresses, it becomes increasingly difficult for afflictedsubjects to organize activities, behave appropriately, and care foroneself. There are currently no treatments to slow or stop theprogression of the disease.

Dementia with Lewy Bodies

Dementia with Lewy bodies (DLB) is a type of dementia that is related toPD. The hallmark of this disease is the presence of alpha synucleinaggregates in brains of afflicted subjects. These patients experiencePD-like symptoms including hunched posture, rigid muscles, a shufflingwalk and trouble initiating movement as well as changes in reasoning andthinking, memory loss (but less significantly than AD). Since Lewybodies are also present in PD, these two diseases may be linked to thesame underlying abnormalities in how the brain processes the proteinalpha-synuclein. Furthermore, similar to microglia-relatedneuroinflammation is present in brains of subjects with DLB, althoughthis pathological feature occurs more extensively (lannaccone et al,Parkinsonism Relat. Disord. 2013 19: 47-52).

Motor Neuron Diseases

Motor neuron diseases (MND), are neurological disorders, similar to ALS,that selectively affect motor neurons, the cells that control voluntarymuscle activity including speaking, walking, swallowing, and locomotoractivities. There is no effective treatment for MND. They areneurodegenerative in nature, and cause progressive disability and death.Furthermore, a specific pathway called progranulin can triggerinflammatory activation of microglia in an animal model of MND andgenetic ablation of this pathway can delay disease progression (Philipset al J. Neuropathol Exp Neurol. 2010 69:1191-200).

Demyelinating Diseases

Demyelinating diseases such as Guillain-Barré syndrome and multiplesclerosis (MS) are degenerative disorders in which in which the myelinsheath of neurons is compromised. This damage impairs signalconductivity in the affected nerves, causing deficiency in sensation,movement, cognition, or other functions. There is no cure for thesediseases. Its most well-known form is MS, a disease in which thecellular subsets of the immune system have been implicated. Forinstance, on-going demyelination is often associated with infiltrationof T cells and macrophages from the circulation as well as inflammatoryactivation of microglia (Kutzelnigg et al. Handb. Clin. Neurol. 2014,122:15-58).

There is a need for an improved method for treating neurodegenerativediseases. The method should be effective and well tolerated.

BRIEF DESCRIPTION OF THE DRAWINGS

WT=wild-type animal, Veh=animals treated with vehicle, MPTP-Ctrl=animalstreated with MPTP and 0.5% methylcellulose, MPTP-ZLN=animals treatedwith MPTP and ZLN005, STZ-Ctrl=animals treated with STZ and 0.5%methylcellulose, STZ-ZLN=animals treated with STZ and ZLN005,5XFAD-Ctrl=AD transgenic animals treated with 0.5% methylcellulose,5XFAD-ZLN=AD transgenic animals treated with ZLN005, ALS-Ctr1=ALStransgenic animals treated with 0.5% methylcellulose, ALS-ZLN=ALStransgenic animals treated with ZLN005.

FF=Ppargc1a^(LoxP/LoxP) mice, Cre=Ppargc1a^(LoxP/LoxP)Cx3cr1^(CreER)mice

FIG. 1 shows the survival rate as a percentage of Cre animals and FFanimals within 30 hours after MPTP induction.

FIG. 2 shows that Ppargc1a activator ZLN005 increases expression ofgenes Pgc1a (Ppargc1a), Tfam, Nrf2, Ucp3, Ant, Sod1, Sod2 andupregulates tyrosine hydroxylase (Th).

FIG. 3 shows the Pgc1a (Ppargc1a) protein expression in microglia inanimals treated with Veh, MPTP-Ctrl, and (MPTP-ZLN).

FIG. 4 shows the glucose transporter Slc2a1 levels and lactic acidlevels in animals treated with Veh, MPTP-Ctrl, and MPTP-ZLN.

FIG. 5 shows immunohistochemical analysis of dopaminergic neurons in thesubstantia nigra of animals treated with Veh, MPTP-Ctrl, and MPTP-ZLN.

FIG. 6 shows TNF-α levels secreted by microglia in Cre animals and FFanimals, treated with Veh, MPTP-Ctrl, and MPTP-ZLN.

FIG. 7 shows weights (g) of shredded nestlets by Cre animals and FFanimals, treated with Veh, MPTP-Ctrl, and MPTP-ZLN.

FIG. 8 shows latency of fall (seconds) of Cre animals and FF animals,treated with Veh, MPTP-Ctrl, and MPTP-ZLN.

FIG. 9 shows relative expression level of several genes in animalstreated with Veh, STZ-Ctrl, and STZ-ZLN.

FIG. 10 shows % of microglia that express TNF-α⁺(A), %ThioltrackerViolet^(hi) (B), and % MitotrackerRed^(hi) (C) in Veh,STZ-Ctrl, and STZ-ZLN.

FIG. 11 shows mean disease scores of STZ-Ctrl and STZ-ZLN mice.

FIG. 12 shows % of microglia that express IL1 (A) and TNFα (B), in WT,5XFAD-Ctrl, and 5XFAD-ZLN.

FIG. 13 shows % of microglia that express Mitotracker Green^(hi) (A),and % microglia that had taken up 2-NBDG (B), in WT, 5XFAD-Ctrl, and5XFAD-ZLN.

FIG. 14 shows % blood monocytes over circulating immune cells in WT,5XFAD-Ctrl, and 5XFAD-ZLN.

FIG. 15 shows nest building activities (g) in WT, 5XFAD-Ctrl, and5XFAD-ZLN.

FIG. 16 shows % of brain perivascular macrophages that express iNOS,IL6, and TNFα in WT, ALS-Ctrl, and ALS-ZLN.

FIGS. 17A-B show latency of fall (seconds) of ALS transgenic animals,treated with 0.5% methylcellulose (Ctrl) or ZLN, at a constant speed(FIG. 17A) and at an accelerating speed (FIG. 17B) in a wheel-runningtest.

FIG. 18 shows % survival vs. time after 100 days in ALS transgenicanimals treated with 0.5% methylcellulose (Ctrl) or ZLN005. Animals weretreated 3 times a week starting at 5, 10, and 15 weeks of age.

FIG. 19 shows % of brain perivascular macrophages among total brainimmune cells in the brain in WT, ALS-Ctrl, and ALS-ZLN.

FIG. 20 shows % of brain perivascular macrophages that have taken up aglucose analog 2-NBDG in WT, ALS-Ctrl, and ALS-ZLN.

FIGS. 21A and 21B show % of total monocytes and % of Ly6C+ inflammatorymonocytes among circulating immune cells, in WT, ALS-Ctrl, and ALS-ZLN.FIG. 21C shows the serum TNF-α levels in WT, ALS-Ctrl, and ALS-ZLN.

FIG. 22 shows latency of fall (seconds) of HD transgenic animals treatedwith 0.5% methylcellulose (Ctrl) or ZLN005.

FIGS. 23A and 23B show latency of fall (seconds) in FF and Cre mice.FF=Ppargc1a^(LoxP/LoxP) mice on DLB transgenic background,Cre=Ppargc1a^(LoxP/LoxP)Cx3cr1^(CreER) mice on DLB transgenicbackground.

FIGS. 24A and 24B show latency of fall (seconds) of DLB transgenicanimals treated with 0.5% methylcellulose (Ctrl) or ZLN005.

DETAILED DESCRIPTION OF THE INVENTION

Inflammatory responses in the brain, which can be demonstrated bychanges in the properties of microglia, a cell type that is located onlyin the brain, are a common feature of human neurodegenerative diseases(Alzheimers Res Ther., 7(1):56. doi: 10.1186/s13195-015-0139-9, 2015).Yong (The Neuroscientist, 16:408-420, 2010) reports that inflammation ofthe central nervous system (CNS) (neuroinflammation) is a feature of allneurological disorders; microglia activation is a cause of thisinflammatory response and microglia-mediated neuroinflammation ispresent in all neurodegenerative disorders.

The inventors have discovered that Ppargc1a, a pleotropic regulator ofcellular metabolism in many cell types, is an important regulator of allneurodegenerative diseases, in which neuroinflammation is mediated bymicroglia. The inventors have discovered a connection between Ppargc1activation in microglia and its effect on the cognitive and motorfunctions of the whole organism. The inventors have discovered thatPpargc1a expression is decreased in humans and animal models withneurodegenerative diseases. The inventors have shown that Ppargc1asignaling in microglia is an important regulator of motor dysfunctionand behavioral dysfunction in animal models and provided evidence thattargeting Ppargc1a with its activator improves motor/behaviordysfunction in neurodegenerative diseases.

The present invention is directed to a method for treatingneurodegenerative diseases. The method comprises the step ofadministering an effective amount of a Ppargc1a activator to a subjectsuffering from a neurodegenerative disease.

The inventors have demonstrated that2-(4-tert-butylphenyl)-1H-benzimidazole,2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, CAS Number 49671-76-3,also known as ZLN005, is an effective Ppargc1a activator. ZLN005 canpenetrate the blood-brain barrier to activate the Ppargc1a pathway inmicroglia, and is effective for treating neurodegenerative diseases.

The chemical structure of ZLN005 is shown below.

Neurodegenerative diseases, as used herein, refers to diseases thatoccur as a result of neurodegenerative processes, i.e., progressive lossof structure or function of neurons and/or death of neurons.Neurodegenerative diseases are incurable and debilitating, and patientstypically have problems with movement (ataxias) and/or mentalfunctioning (dementias). Neurodegenerative diseases include ALS, AD, PD,HD, frontotemporal degeneration disease, dementia with Lewy bodies,motor neuron diseases, demyelinating diseases (such as Guillain-Barrésyndrome and multiple sclerosis), prion disease, spinocerebellar ataxia,and spinal muscular atrophy.

The inventors have discovered that activation of the Ppargc1a pathway inmicroglia by ZLN005 can suppress microglia-mediated inflammatoryresponses. Deletion of Ppargc1a specifically in microglia acceleratesneuropathological development in transgenic animal models PD (MPTP) anddementia with Lewy bodies (SNCA*A53T). Furthermore in transgenic animalmodels of PD (MPTP), AD (5XFAD and icv-STZ), HI) (R6/2), ALS(SOD1*G93A), and dementia with Lewy bodies (SNCA*A53T); treatment withZLN005 significantly alleviates behavioral dysfunction. Collectively,ZLN005 represents a treatment for all neurodegenerative disorders inwhich microglia-mediated neuroinflammation contributes to the diseasedevelopment.

ALS

Circulating monocytes from the blood give rise to brain perivascularmacrophages, which reside just outside the vascular basement membrane.They are the main antigen-presenting cells of the CNS, thus playing animportant role in immune reactions involving the brain. Along withmicroglia, brain perivascular macrophages are the earliest macrophagesfrom peripheral tissues that response to brain injuries. Their locationat the interface between brain parenchyma and the vascular system andtheir continuous circulation in and out of blood vessels suits themideally for this function.

The inventors have discovered that brain perivascular macrophages in ALStransgenic mice exhibited an inflammatory phenotype, evidenced by asignificant increase in iNOS production. By administering ZLN005 tothese animals, iNOS production in the brain perivascular macrophagesdecreased and neuroinflammation was suppressed.

The inventors have provided evidence that ALS transgenic mice treatedwith ZLN005 had improved motor skills compared with untreated ALStransgenic mice.

The inventors have also shown that ALS transgenic mice exhibited hindlimb paralysis at approximately 100 days and died shortly after. Byadministering ZLN005 to these animals, the onset of hind limb paralysiswas delayed and the survival rate increased.

AD

Administration of streptozocin (STZ) by intracerebral injection to miceand non-human primates is a well-established animal model of thesporadic form of AD (Arabpoor et al Adv Biomed Res, 1:50, 2012)

The inventors have discovered that administering ZLN005 to theSTZ-treated animal resulted in increased expression of genes involved inPpargc1a signaling, mitochondrial metabolism, and anti-oxidative defensein the brain

The principal chemical constituent of the amyloid plaques and amyloidangiopathy characteristic of AD is an approximately 4.2 KD protein ofβ-amyloid peptide. STZ-treated animals significantly increase theexpression of β-amyloid peptide. By administering ZLN005, the expressionof genes involved in β-amyloid generation in the brains of STZ-treatedanimals was decreased to normal levels.

The inventors have shown that microglia in STZ-treated mice exhibited aninflammatory phenotype, evidenced by a significant increase in TNF-αproduction. Administering ZLN005 to the STZ-treated mice resulted insuppression of TNF-α production in the microglia cells and suppressionof the microglia-mediated neuroinflammation. The inventors alsodiscovered that ZLN005 modulated metabolic dysfunction in microgliainduced by STZ, as evidenced by enhanced glycolysis, mitochondrialpotential, and glutathione production in microglia isolated fromSTZ-treated animals and treated by ZLN005.

STZ-treated mice exhibit several signs and symptoms of behavioraldysfunction and systemic inflammation including bleeding from the nose,eyes, ears, paralysis of hands and feet (Arabpoor et al Adv Biomed Res,1:50, 2012). Administering ZLN005 to the STZ-treated mice resulted in asignificant reduction in the disease severity.

PD

Administration of the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a sub-lethal doseto mice and non-human primates results in a neurodegenerative diseasethat is similar to PD in its pathology and symptoms, and this wellestablished animal model has been widely used for drug screening(Blandini et al, FEBS J, 279:1156-66, 2012).

The inventors have generated microglia specific knockout of Ppargc1a, inwhich Ppargc1a signaling is absent in these cells and not in other cellsof the brain such as neurons. When PD was induced with MPTP in wild-typeand microglia specific knockout animals, the knockout animals hadsignificantly more severe motor impairment, indicating that Ppargc1asignaling in microglia regulates behavioral dysfunction.

By analyzing the expression of specific genes in the brain, theinventors have shown that MPTP markedly inhibited Ppargc1a signaling andthe anti-oxidant defense system. By administering ZLN005 to theMPTP-treated animal, the expression of genes involved in Ppargc1asignaling, anti-oxidative stress, and dopamine synthesis in the brainsof those animals was increased. In addition, since ZLN005 wasadministered orally, it penetrated the blood-brain barrier as indicatedby its activation of the Ppargc1a pathway in microglia.

The inventors have shown that microglia in MPTP-treated mice exhibitedan inflammatory phenotype, evidenced by a significant increase in TNF-αproduction and a decrease in mitochondrial biogenesis. AdministeringZLN005 to the MPTP-treated mice resulted in decreased TNF-α productionin the microglia cells and suppression of microglia-mediatedneuroinflammation.

At the organismal level, MPTP-treated mice exhibit profound loss of finemotor skills and behavioral dysfunctions. The inventors have shown thatby administering ZLN005 to the MPTP-treated mice, the motor skills ofthose mice were improved.

HD

Targeting Ppargc1a with its activator, ZLN005, ameliorates motordysfunction in Huntington's disease (HD). The inventors have providedevidence that targeting Ppargc1a with ZLN005 improved motor skills in HDtransgenic mice. The inventors have shown that HD transgenic micetreated with ZLN005 exhibited improved motor skills, as indicated byincreases in their latency to fall, compared with untreated HDtransgenic mice.

Dementia with Lewy Bodies

Targeting Ppargc1a with its activator ZLN005 ameliorates motordysfunction in dementia with Lewy bodies. The inventors have shown thatmicroglia-specific deletion in transgenic DLB animals caused furtherdeterioration of motor function in the animals. The inventors have alsodemonstrated that Ppargc1a activator, ZLN005, improved motor skills inDLB transgenic animals.

Frontotemporal Degeneration

Frontotemporal degeneration, also called frontotemporal dementia (FTD)is a disease that is closely related to ALS in which progressivedegeneration occurs in the frontal and temporal lobes of the brain.

By suppressing microglia-mediated inflammation, ZLN005 improves motorskills in FTD transgenic mice and increases their survival rate.

Motor Neuron Diseases

Motor neuron diseases are neurodegenerative disorders, similar to ALS,that selectively affect motor neurons. Microglia-mediated inflammationis a key factor for development factor for motor neuron diseases. Bysuppressing microglia-mediated inflammation, ZLN005 slows down and haltsdisease development.

Demyelinating Diseases

ZLN005 is effective in treating demyelinating diseases by reducing theinflammatory activation of microglia, which might be more susceptible toinflammatory stimuli in demyelinating diseases such as multiplesclerosis. By suppressing metabolic dysregulation and subsequentinflammatory transformation of microglia, ZLN005 promotes myelin repairand regeneration.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprisingone or more pharmaceutically acceptable carriers and an active compoundof 2-(4-tert-butylphenyl)-1H-benzimidazole,2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole (ZLN005), or apharmaceutically acceptable salt, or a solvate thereof. The activecompound or its pharmaceutically acceptable salt or solvate in thepharmaceutical compositions in general is in an amount of about 0.01-20%(w/w) for a topical formulation; about 0.1-5% for an injectableformulation, 0.1-5% for a patch formulation, about 1-90% for a tabletformulation, and 1-100% for a capsule formulation.

In one embodiment, the pharmaceutical composition can be in a dosageform such as tablets, capsules, granules, fine granules, powders,syrups, suppositories, injectable solutions, patches, or the like. Inanother embodiment, the pharmaceutical composition can be an aerosolsuspension of respirable particles comprising the active compound, whichthe subject inhales. The respirable particles can be liquid or solid,with a particle size sufficiently small to pass through the mouth andlarynx upon inhalation. In general, particles having a size of about 1to 10 microns, preferably 1-5 microns, are considered respirable.

In another embodiment, the active compound is incorporated into anyacceptable carrier, including creams, gels, lotions or other types ofsuspensions that can stabilize the active compound and deliver it to theaffected area by topical applications. The above pharmaceuticalcomposition can be prepared by conventional methods.

Pharmaceutically acceptable carriers, which are inactive ingredients,can be selected by those skilled in the art using conventional criteria.Pharmaceutically acceptable carriers include, but are not limited to,non-aqueous based solutions, suspensions, emulsions, microemulsions,micellar solutions, gels, and ointments. The pharmaceutically acceptablecarriers may also contain ingredients that include, but are not limitedto, saline and aqueous electrolyte solutions; ionic and nonionic osmoticagents such as sodium chloride, potassium chloride, glycerol, anddextrose; pH adjusters and buffers such as salts of hydroxide,phosphate, citrate, acetate, borate; and trolamine; antioxidants such assalts, acids and/or bases of bisulfite, sulfite, metabisulfite,thiosulfite, ascorbic acid, acetyl cysteine, cysteine, glutathione,butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, andascorbyl palmitate; surfactants such as lecithin, phospholipids,including but not limited to phosphatidylcholine,phosphatidylethanolamine and phosphatidyl inositiol; poloxamers andpoloxamines, polysorbates such as polysorbate 80, polysorbate 60, andpolysorbate 20, polyethers such as polyethylene glycols andpolypropylene glycols; polyvinyls such as polyvinyl alcohol andpovidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose andhydroxypropyl methylcellulose and their salts; petroleum derivativessuch as mineral oil and white petrolatum; fats such as lanolin, peanutoil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers ofacrylic acid such as carboxypolymethylene gel, and hydrophobicallymodified cross-linked acrylate copolymer; polysaccharides such asdextrans and glycosaminoglycans such as sodium hyaluronate. Suchpharmaceutically acceptable carriers may be preserved against bacterialcontamination using well-known preservatives, these include, but are notlimited to, benzalkonium chloride, ethylenediaminetetraacetic acid andits salts, benzethonium chloride, chlorhexidine, chlorobutanol,methylparaben, thimerosal, and phenylethyl alcohol, or may be formulatedas a non-preserved formulation for either single or multiple use.

For example, a tablet formulation or a capsule formulation of the activecompound may contain other excipients that have no bioactivity and noreaction with the active compound. Excipients of a tablet or a capsulemay include fillers, binders, lubricants and glidants, disintegrators,wetting agents, and release rate modifiers. Binders promote the adhesionof particles of the formulation and are important for a tabletformulation. Examples of excipients of a tablet or a capsule include,but not limited to, carboxymethylcellulose, cellulose, ethylcellulose,hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch,tragacanth gum, gelatin, magnesium stearate, titanium dioxide,poly(acrylic acid), and polyvinylpyrrolidone. For example, a tabletformulation may contain inactive ingredients such as colloidal silicondioxide, crospovidone, hypromellose, magnesium stearate,microcrystalline cellulose, polyethylene glycol, sodium starchglycolate, and/or titanium dioxide. A capsule formulation may containinactive ingredients such as gelatin, magnesium stearate, and/ortitanium dioxide.

For example, a patch formulation of the active compound may comprisesome inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminumaminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin,methylparaben, polysorbate 80, povidone, propylene glycol,propylparaben, sodium carboxymethylcellulose, sodium polyacrylate,tartaric acid, titanium dioxide, and purified water. A patch formulationmay also contain skin permeability enhancer such as lactate esters(e.g., lauryl lactate) or diethylene glycol monoethyl ether.

Topical formulations including the active compound can be in a form ofgel, cream, lotion, liquid, emulsion, ointment, spray, solution, andsuspension. The inactive ingredients in the topical formulations forexample include, but not limited to, diethylene glycol monoethyl ether(emollient/permeation enhancer), DMSO (solubility enhancer), siliconeelastomer (rheology/texture modifier), caprylic/capric triglyceride,(emollient), octisalate, (emollient/UV filter), silicone fluid(emollient/diluent), squalene (emollient), sunflower oil (emollient),and silicone dioxide (thickening agent).

Method of Use

The present invention is directed to a method of treatingneurodegenerative diseases. The method comprises the step ofadministering to a subject suffering from a neurodegenerative disease aneffective amount of 2-(4-tert-butylphenyl)-1H-benzimidazole,2-[4-(1,1-dimethylethyl)phenyl]-1H-benzimidazole, for treating theneurodegenerative disease. “An effective amount,” as used herein, is theamount effective to treat the neurodegenerative disease by amelioratingthe pathological condition or reducing the symptoms of the disease.

In one embodiment, the neurodegenerative disease is ALS and the methodreduces or alleviates motor dysfunction or behavioral dysfunction in anALS patient. For example, the method improves early symptoms such asdifficulty in walking or doing normal daily activities; weakness inlegs, feet, ankles, or hand; tripping or clumsiness; slurring of speechor trouble swallowing; and muscle cramps and twitching in the arms,shoulders and tongue. The method may also improve later symptoms such asdifficulty in breathing. In another important embodiment, the methodimproves survival rate and length of survival.

In one embodiment, the neurodegenerative disease is AD and the methodreduces or alleviates the disease symptoms and improves the cognitiveand motor functions. For example, the method improves confusion,irritability, aggression, mood swings, trouble with language, and/orlong-term memory loss in a patient. The method may also slow down thedisease progression.

In one embodiment, the neurodegenerative disease is PD and the methodreduces or alleviates motor dysfunction or behavioral dysfunction in apatient. For example, the method improves movement-related symptoms suchas shaking, rigidity, slowness of movement, and difficulty with finemotor skills, walking, and gait.

In one embodiment, the neurodegenerative disease is HD and the methodreduces or alleviates motor dysfunction in a patient. For example, themethod improves involuntary and/or voluntary movement-related symptomssuch as involuntary jerking or writhing movements (chorea); muscleproblems (e.g., rigidity or muscle contracture (dystonia)); slow orabnormal eye movements; impaired gait, posture and balance; difficultywith the physical production of speech or swallowing.

In one embodiment, the neurodegenerative disease is dementia with Lewybodies (DLB) and the method reduces or alleviates motor dysfunction andcognitive decline in a patient. For example, the method improves PD-likesymptoms such as motor coordination, difficulties with walking andswallowing, inability to maintain normal postures, rigidity as well asloss of memory and decline in thinking and reasoning. The method mayalso halt or slow down disease progression.

In one embodiment, the neurodegenerative disease is frontotemporaldegeneration (FTD) and the method reduces or alleviates the diseasesymptoms that are associated with language skills and socialinteractions. For example, the method improves abilities to speakcoherently, to organize thoughts and daily activities, to interactnormally in social settings and alleviates symptoms of disinhibition,loss of sympathy and empathy, lack of executive control, hyperorality,and apathy. The method may also halt or slow down disease progression.

In one embodiment, the neurodegenerative disease is a motor neurondisease (MND) and the method reduces or alleviates motor dysfunction aswell as improves survival rate and length of survival of patients withthese diseases. For example, the method improves movement-relatedsymptoms such as troubles with walking, maintaining normal gait,controlling balance, difficulties with fine motor coordination, slownessof movement, swallowing, and breathing.

In one embodiment, the neurodegenerative disease is a demyelinatingdisease such as Guillain-Barré syndrome or multiple sclerosis (MS) andthe method reduces or alleviates behavioral dysfunction and cognitiveimpairment in patients with these diseases. For example, the methodimproves early symptoms such as blurred vision, tingling sensation,numbness and weakness in limbs, lack of coordination. The method mayalso improve advanced symptoms such as difficulty in walking, tremors,muscle spasms, paralysis, troubling articulating thoughts and speaking.The method may also improve survival rate and length of survival.

The pharmaceutical composition of the present invention can be appliedby systemic administration or local administration. Systemicadministration includes, but is not limited to oral, parenteral (such asintravenous, intramuscular, subcutaneous or rectal), and inhaledadministration. In systemic administration, the active compound firstreaches plasma and then distributes into target tissues. Oraladministration is a preferred route of administration for the presentinvention. Local administration includes topical administration.

Dosing of the composition can vary based on the extent of the injury andeach patient's individual response. For systemic administration, plasmaconcentrations of the active compound delivered can vary; but aregenerally 1×10⁻¹⁰-1×10⁻⁴ moles/liter, and preferably 1×10⁻⁸-1×10⁻⁵moles/liter.

In one embodiment, the pharmaceutical composition is administratedorally to a subject. The dosage for oral administration is generally0.1-100, 0.1-20, or 1-50 mg/kg/day, depending on the subject's age andcondition. For example, the dosage for oral administration is 0.1-10,0.5-10, 1-10, 1-5, or 5-50 mg/kg/day for a human subject. In oneembodiment, the active compound can be applied orally to a human subjectat 1-100, 10-50, 20-1000, 20-500, 100-800 sage, or 200-600 mg/dosage,1-4 times a day, depends on the patient's age and condition.

In one embodiment, the pharmaceutical composition is administratedintravenously to a subject. The dosage for intravenous bolus injectionor intravenous infusion is generally 0.03 to 5 or 0.03 to 1 mg/kg/day.

In one embodiment, the pharmaceutical composition is administratedsubcutaneously to the subject. The dosage for subcutaneousadministration is generally 0.3-20, 0.3-3, or 0.1-1 mg/kg/day.

In one embodiment, the composition is applied topically to an area andrubbed into it. The composition is topically applied at least 1 or 2times a day, or 3 to 4 times per day, depending on the medical issue andthe disease pathology. In general, the topical composition comprisesabout 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5%(w/w) of the active compound. Typically 0.2-10 mL of the topicalcomposition is applied to the individual per dose. The active compoundpasses through skin and is delivered to the site of discomfort.

Those of skill in the art will recognize that a wide variety of deliverymechanisms are also suitable for the present invention.

The present invention is useful in treating a mammal subject, such ashumans, horses, dogs and cats. The present invention is particularlyuseful in treating humans.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES

All animal studies were conducted under protocols approved by APLAC fromStanford University. 8-10 week old C57BL6/J mice were used in allexperiments. Data were presented as mean±SEM. Two-tailed Student'st-test, two-way ANOVA, and log rank test were used for statisticalanalyses. A p value of <0.05 was considered to be statisticallysignificant.

A. Examples 1-8 Relate to PD.

Veh=animals treated with PBS as vehicle, MPTP-Ctrl=animals treated withMPTP and 0.5% methylcellulose, MPTP-ZLN=animals treated with MPTP andZLN005. FF=Ppargc1a^(LoxP/LoxP) mice,Cre=Ppargc1a^(LoxP/LoxP)Cx3cr1^(CreER) mice.

Example 1. Ppargc1a Deletion in Microglia Accelerates MPTP-InducedMortality

Animals with microglia-specific deletion of Ppargc1a were generated bycrossing mice harboring the foxed allele of Ppargc1a(Ppargc1a^(LoxP/LoxP)) with those expressing Tamoxifen inducible Crerecombinase under the control of Cx3cr1 promoter (Cx3cr1^(CreER)). Toinduce Cre-mediated deletion of Ppargc1a in Cx3cr1 expressing cells,Tamoxifen (Sigma) in 200 μl corn oil (50 mg/ml, Sigma) was administeredto 3 weeks old (Ppargc1a^(LoxP/LoxP)Cx3cr1^(CreER)) mice twice at48-hour intervals (Wolf et al Front Cell Neurosci 2013, 18; 7:26. doi:10.3389/fncel.2013.00026). Littermates carrying the floxed allele ofPpargc1a alleles but lacking expression of Cre recombinase(Ppargc1a^(LoxP/LoxP)) were used as controls. Animals were rested foranother 5-6 weeks before MPTP was administered.

To induce symptoms of PD, MPTP (20 mg/kg) was administeredintraperitoneally in sterile PBS 4 times at 2-hour intervals on day 1.Control animals received a similar volume of PBS. After MPTP induction,7 out of 12 Cre animals died within 30 hours, while 3 out of 16 FFanimals died within 30 hours. The results are shown in FIG. 1. Log-ranktest was used for statistical analysis. The results show that Ppargc1adeletion in microglia accelerates MPTP-induced mortality.

Example 2. Ppargc1a Activator ZLN005 Upregulates Expression ofParticular Genes in the Brain

Ppargc1a, an inducer of mitochondrial biogenesis, is widely expressed incells throughout the body. Ppargc1a activator ZLN005 (25 mg/kg, Sigma)was administered orally once a day starting 30 minutes after MPTPadministration on Day 1 (when animals exhibited PD-like symptoms) for 3consecutive days in 0.5% methylcellulose (Sigma).

For gene expression studies, animals were sacrificed on Day 4, 24 hoursafter the 3rd oral dosage of ZLN005, and PBS-perfused brain tissues wereprocessed for RNA isolation, cDNA synthesis, and real-time quantitativePCR (Invitrogen). The results are summarized in FIG. 2. The results showthat the Ppargc1a activator, ZLN005, increases expression of genesinvolved in Ppargc1a signaling (Pgc1a), mitochondrial genes (Tfam, Nrf2,Ucp3—downstream targets of Ppargc1a), and anti-oxidative stress genes(Ant, Sod1, Sod2) in the brains of MPTP-treated animals. There was alsoa 15% upregulation of tyrosine hydroxylase (Th), the enzyme that iscritical for dopamine synthesis in the brain. These results indicatethat ZLN005 penetrated the blood-brain barrier and activated thePpargc1a pathway in the brain. Unpaired t-tests were used forstatistical analyses.

Example 3. Ppargc1a Activator ZLN005 Upregulates Ppargc1a ProteinExpression in Microglia

For protein expression studies, animals were sacrificed on Day 4, 24hours after the 3rd oral dosage of ZLN005 and PBS-perfused brain tissueswere digested with Collagenase IV, processed for microglia isolation byflow cytometry (Ginhoux et al Science, 330:841-5, 2010), and immunoblotanalysis of Ppargc1a expression. The results are summarized in FIG. 3.The results show that MPTP administration suppressed Ppargc1a proteinexpression in microglia, and that ZLN005 penetrated the blood-brainbarrier and enhanced Ppargc1a protein expression in microglia inMPTP-treated animals.

Example 4. Ppargc1a Activator ZLN005 Reverses Microglial MetabolicReprogramming Induced by MPTP

For metabolic phenotyping studies, animals were sacrificed on Day 4, 24hours after the third oral dosage of ZLN005 and PBS-perfused braintissues were processed for microglia isolation and flow cytometryanalysis of glucose metabolism in microglia.

For measuring microglial expression of glucose transporter Slc2a1,MPTP-ZLN (n=10), MPTP-Ctrl (n=14), and Veh animals (n=14) weresacrificed. Brain microglia were phenotyped with antibody directedagainst glucose transporter Slc2a1 (RnD) for flow cytometric acquisition(LSRII, BD) and analysis (FlowJo).

For measuring microglial glycolysis, MPTP-ZLN (n=8), MPTP-Ctrl (n=6),and Veh animals (n=6) were sacrificed. Microglia were sorted by flowcytometry and subjected to lactic acid production assays ex vivo (CaymanChem) for glycolysis measurement.

The results are summarized in FIG. 4. Y-axis represents Slc2a1expression in median fluorescence units (MFI, A) and lactic acidproduction in micromolar units (mM, B). The results show that microgliain MPTP-treated mice exhibited a glycolytic activation phenotype,measured by increases in glucose transporter Slc2a1 expression (A) andlactic acid production (B), in non-treated MPT-intoxicated animals whencompared with Veh mice. The results also show that by administeringZLN005 to MPTP-treated animals, glucose transporter expression andlactic acid production in microglia of these treated animals decreased,and thus their metabolic dysfunction was corrected. ANOVA was used forstatistical analyses.

Example 5. Ppargc1a Activator ZLN005 Reverses Dopaminergic Degenerationin MPTP-Treated Animals

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally oncea day starting 30 minutes after MPTP administration on Day 1 for 7consecutive days in 0.5% methylcellulose (Sigma). For protein expressionstudies, animals were sacrificed on Day 8, 24 hours after the 7th oraldosage of ZLN005, and paraformaldehyde-perfused brain tissues wereprocessed for immunohistochemical analysis of dopaminergic neurons inthe substantia nigra.

One representative picture of each of Veh (n=3), MPTP-Ctrl (n=5), andMPTP-ZLN (n=5) animals is shown in FIG. 5. The brown staining representstyrosine hydroxylase expression in dopaminergic neurons of thesubstantia nigra. The results show that MPTP administration led to adepletion of these neurons, which was reversed by treatment with ZLN005.

Example 6. ZLN005 Suppresses TNF-α Production in a Microglial Ppargc1aDependent Manner

Animals with microglia-specific deletion of Ppargc1a and controls weregenerated as described in Example 1. For flow cytometry studies, animalswere sacrificed on Day 4, 24 hours after the 3rd oral dosage of ZLN005.PBS-perfused brain tissues of sacrificed animals were digested withCollagenase IV and isolated by flow cytometry.

For measuring microglial production of TNF-α, MPTP-ZLN (n=5), MPTP-Ctrl(n=10) and Veh (n=6) animals were sacrificed. Isolated microglia weresubjected to a 2-hour ex vivo TNF-α production assay. Supernatantsamples were collected for TNF-α measurement with CBA technology (BD).

The results are summarized in FIG. 6. The results show that MPTPadministration induced TNF-α secretion by microglia in FF animals andthis induction of TNF-α production was significantly higher in Creanimals. Furthermore, ZLN005 suppressed TNF-α production in microgliaisolated from MPTP-treated FF animals but failed to exert itsanti-inflammatory effects on microglia isolated from MPTP-treated Creanimals, which had Cre-mediated deletion of Ppargc1a in Cx3cr1expressing microglia. These results indicate that ZLN005 suppressesexpression of the inflammatory cytokine TNF-α in microglia via itsactivation of microglia specific Ppargc1a. Unpaired t-tests were usedfor statistical analyses.

Example 7. ZLN005 Improves Fine Motor Skills in a Microglial Ppargc1aDependent Manner

Impaired nest-building skill has been widely used as one of the mostreliable indication of motor dysfunction in the MPTP model of PD(Sedelis et al, Behav Brain Res, 125:109-25, 2001). In this test,animals are given cotton pads to be used as nestling, and are tested fortheir abilities to tear off the cotton pads into small pieces to build anest; these abilities require fine motor coordination.

Animals with microglia-specific deletion of Ppargc1a and controls weregenerated as described in Example 1. To induce symptoms of PD, MPTP wasadministered intraperitoneally in sterile PBS 4 times at 2-hourintervals on day 1. ZLN005 was administered once 30 minutes after thefirst dose of MPTP, when animals exhibited PD symptoms. Control animalsreceived a similar volume of PBS. Immediately after the last dosage ofMPTP, each animal was put in one cage and given two cotton pads to beused in nestling; the appearance of the cotton pads was evaluated 16hours later.

The results are shown in FIG. 7 as weights of shredded nestlets (n=9-10animals per treatment group). Animals who received PBS exhibited normalnest-building activity, while MPTP-treated mice failed to tear off thecotton pads into small pieces to make their nest. The results show thatwith one dose of ZLN005 at 25 mg/kg after disease induction,MPTP-treated animals exhibited marked improvement in their fine motorskills. MPTP-ZLN animals generated more cotton-debris in comparison tothe MPTP-Ctrl group. Further, this effect was present only in FF animalsbut not in Cre animals, which had Cre-mediated deletion of Ppargc1a inCx3cr1 expressing microglia. The results show that ZLN005 improves finemotor skills in MPTP-treated animals, and this beneficial effect ofZLN005 on nest building activity requires microglia specific Ppargc1a.Unpaired t-tests were used for statistical analyses.

Example 8. ZLN005 Improves Motor Coordination in a Microglial Ppargc1aDependent Manner

The wheel-running test has been widely used as one of the most reliablemeasurements of behavioral dysfunction in animal models ofneurodegeneration (Sedelis et al, Behav Brain Res, 125:109-25, 2001). Inthis study, animals were trained to run on a treadmill at specific speedand training duration before undergoing a formal test of motor skills.Motor performance of animals was evaluated by the time (seconds) thatthey remained running on the treadmill, which required motorcoordination and strength. Longer running time on treadmill suggestsenhanced motor skills.

Animals with microglia-specific deletion of Ppargc1a were generated asdescribed in Example 1. At 7 weeks of age, these animals were subjectedto 1.5 weeks of training on a treadmill at a constant speed 10 rpm(rotations per minute) and then 1.5 weeks of training at an acceleratingspeed from 5-15 rpm. After the training period at 10 weeks of age,animals were treated with MPTP and tested for motor performance at anaccelerating speed from 5-15 rpm.

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally tothe animals once a day starting 30 minutes after MPTP administration onDay 1 for 7 consecutive days in 0.5% methylcellulose (Sigma) and theanimals were trained on a treadmill at an accelerating speed from Day 2to Day 7 and tested on Day 8. The results of latency to fall in secondsof each group (n=5-21 per group) on Day 8 are shown in FIG. 8.

FIG. 8 shows that MPTP treatment impaired wheel running time in FFanimals and ZLN005 treated mice performed significantly better thanvehicle treated mice. The ability of ZLN005 to improve wheel-runningskills is not present in Cre animals, which had Cre-mediated deletion ofPpargc1a in Cx3cr1 expressing microglia. These results indicate thatZLN005 improves motor skills in MPTP-treated animals in a microglialPpargc1a dependent manner. Unpaired t-tests were used for statisticalanalyses.

B. Examples 9-15 Relate to AD.

To induce symptoms of AD, STZ (3 mg/kg, Sigma) was administered viaintracerebral injection (Kalafatakis et al, Int J Neurosci, PMID24494726, 2014). This is a well-established model of the sporadic formof AD. Briefly, animals were anesthetized and STZ solution in 5 μlartificial cerebrospinal fluid (Harvard Apparatus) was injected throughthe skull with a 50 μl syringe. Control animals received a similarvolume of artificial cerebrospinal fluid without STZ. The injectionswere performed twice, on Day 1 and Day 3.

Veh=animals treated with artificial cerebrospinal fluid as vehicle inSTZ model, STZ-Ctrl=animals treated with STZ and 0.5% methylcellulose,STZ-ZLN=animals treated with STZ and ZLN005, WT=wild-type animals,5XFAD-Ctrl=transgenic AD animals treated with 0.5% methylcellulose,5XFAD-ZLN=transgenic AD animals treated with ZLN005.

Example 9. Ppargc1a Activator ZLN005 Upregulates Expression ofParticular Genes in the Acute STZ Model of AD

Ppargc1a, which is an activator of mitochondrial biogenesis, is widelyexpressed in cells throughout the body. Ppargc1a activator ZLN005 (25mg/kg, Sigma) was administered orally once on Day 1 in 0.5%methylcellulose (Sigma) immediately before the first dose of STZ.Treatment with ZLN005 was continued on a daily schedule until Day 4.

For gene expression studies, animals were sacrificed on Day 4, andPBS-perfused brain tissues were processed for RNA isolation, cDNAsynthesis and real-time quantitative PCR (Invitrogen).

The results are summarized in FIG. 9. The results show that Ppargc1aactivator ZLN005 increased expression of genes involved in Ppargc1asignaling and antioxidant defense (Tfam, Cytc), and decreased theexpression of genes involved in β-amyloid generation (App and Psen1), inthe brains of STZ-treated-animals (n=10 animal per condition). Unpairedt-tests were used for statistical analyses.

Example 10. Ppargc1a Activator ZLN005 Suppresses TNF-α Production andMetabolic Abnormalities in Microglia in the Acute STZ Model of AD

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally oncein 0.5% methylcellulose (Sigma) immediately before the first dose of STZon Dayl. Treatment with ZLN005 was continued on a daily schedule untilDay 7.

For microglia analysis, animals were sacrificed on Day 7, andPBS-perfused brain tissues were digested with Collagenase IV andprocessed for flow cytometry. Microglia were phenotyped with antibodiesdirected against mouse TNF-α (Biolegend) and metabolic dyesThioltrackerViolet, and MitotrackerRed (Invitrogen) for flow cytometricacquisition (LSRII, BD) and analysis (FlowJo). The results aresummarized in FIG. 10.

The results show that ZLN005 suppressed TNF-α production in microgliaisolated from STZ-Ctrl (n=8) when compared with Veh animals (n=8) (A),which indicates that neuroinflammation was inhibited. The results alsoshow that ZLN005 enhanced (B) the production of glutathione, anantioxidant with neuroprotective properties, and (C) mitochondrialpotential, a marker of functional integrity of mitochondria, inmicroglia isolated from STZ-treated animals (n=4). These resultsindicate that ZLN005 reverses metabolic dysfunction in microglia inducedby STZ. ANOVA was used for statistical analyses.

Example 11. Ppargc1a Activator ZLN005 Decreases Disease Severity in theAcute STZ Model of AD

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally onceon Day 1 in 0.5% methylcellulose (Sigma) immediately before the firstdose of STZ. Treatment with ZLN005 continued on a daily schedule untilDay 4, when the animals were evaluated.

The STZ-ZLN mice (n=14) appeared more active, less lethargic and none ofthe animals were paralyzed, compared with STZ-Ctrl mice (n=15). InSTZ-ZLN mice, 33% of the animals were active, 66% showed evidence oflethargy, and none were paralyzed. In contrast, in STZ-Ctrl mice, only10% were active, 70% were lethargic, and 20% of these animals had hindlimb paralysis. Veh animals receiving intracerebral artificialcerebrospinal fluid exhibited normal behavior.

The inventors designed a disease scoring system based on evidence oftissue inflammation: with scores of 1 (mild inflammation, increasedvascularization/bleeding of internal organs), 2 (moderate inflammation,severe vascularization/bleeding of internal organs), and 3 (severeinflammation, intestinal or stomach swelling). The disease scoringsystem is also based on physical activity with scores of 1 (lethargic,general poverty of movements with signs of lethargy), 2 (inactive, lackof movement for more than 15 consecutive seconds), and 3 (paralysis ofeither front or hind limbs). The disease score presented in the exampleis the total score of the two scoring systems.

The mean disease scores of animals on day 4 in STZ-Ctrl and STZ-ZLN areshown in FIG. 11. STZ-ZLN mice had a significantly lower mean diseasescore (1.6) compared to STZ-Ctrl (2.5), indicating that the diseaseseverity was improved by the ZLN005 treatment. Animals receivingintracerebral artificial cerebrospinal fluid behaved normally and had amean score of 0. Unpaired t-test was used for statistical analysis.

Example 12. Ppargc1a Activator ZLN005 Suppresses Neuroinflammation inMicroglia in AD Transgenic Animals

5XFAD transgenic mice, which are model of familial AD, were purchasedfrom Jackson Laboratories (Oakley et al J Neurosci. 26:10129-40, 2006).These animals overexpress both mutant human APP(695) with the Swedish(K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer'sDisease (FAD) mutations and human PS1 harboring two FAD mutations, M146Land L286V. These transgenic mice rapidly recapitulate major features ofamyloid pathology in AD by 8-10 weeks of age. Microglia abnormalitiesand neuroinflammation are also pronounced within this time window.Subsequently, neurodegeneration and behavioral dysfunction that mimiccognitive and psychiatric symptoms of human AD begin and are pronouncedby 4-5 months of age.

AD transgenic animals were orally treated 3 times a week for 4 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 3 weeks of age.

For studies of microglia, 5XFAD-ZLN (n=10), 5XFAD-Ctrl (n=8) and WT(n=11) animals were sacrificed at 7 weeks of age. PBS-perfused braintissues of sacrificed animals were digested with Collagenase IV andprocessed for flow cytometry. Brain microglia were phenotyped withantibodies directed against mouse IL1 and TNFα (Biolegend) for flowcytometric acquisition (LSRII, BD) and analysis (FlowJo).

The results are summarized in FIG. 12; Y-axis represents % of microgliathat express IL1 (A) and TNFα (B). The results show that microglia in ADtransgenic mice exhibited an inflammatory phenotype, evidenced by asignificant increase in IL1 production in 5XFAD-Ctrl when compared withWT mice. The results also show that by administering ZLN005 to ADtransgenic animals, IL1 and TNFα production in microglia of thesetreated animals decreased and thus neuroinflammation was suppressed.ANOVA was used for statistical analyses.

Example 13. Ppargc1a Activator ZLN005 Suppresses Metabolic Dysfunctionin Microglia in AD Transgenic Animals

AD transgenic animals were orally treated 3 times a week for 4 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 3 weeks of age.

For studies of microglia, 5XFAD-ZLN (n=10) and 5XFAD-Ctrl (n=8) animalsand WT animals (n=11) were sacrificed at 7 weeks of age. PBS-perfusedbrain tissues of sacrificed animals were digested with Collagenase IVand processed for flow cytometry. Brain microglia were phenotyped with2-NBDG and MitotrackerGreen (Invitrogen) for flow cytometric acquisition(LSRII, BD) and analysis (FlowJo).

The results are summarized in FIG. 13; Y-axis represents % of microgliathat highly expressed MitotrackerGreen (A) and had taken up 2-NBDG (B).Mitochondrial respiration and glycolysis are two key energy generatingpathways in living cells. In immune cells like microglia, inflammatorytransformation is associated with upregulation of glucose utilizationand depression of mitochondrial biogenesis and function. The resultsshow that microglia in 5XFAD-Ctrl exhibited a decrease in mitochondrialmass, measured by Mitotracker Green (A), and exhibited a glycolyticactivation phenotype, evidenced by a significant increase in glucoseuptake, measured by 2-NBDG incorporation (B), when compared with WTanimals. The results also show that by treating AD transgenic animalswith ZLN, (A) mitochondrial mass in the cells of 5XFAD-ZLN mice wasenhanced, and (B) glucose uptake in microglia of these treated animalsdecreased, and thus their metabolic dysfunction was corrected. ANOVA wasused for statistical analyses.

Example 14. Ppargc1a Activator ZLN005 Suppresses Blood Monocytosis in ADTransgenic Animals

AD transgenic animals were orally treated 3 times a week for 4 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 3 weeks of age.

For measuring the frequency of CD115+CD11b+ blood monocytes, 5XFAD-ZLN(n=5), 5XFAD-Ctrl (n=4), and WT animals (n=4) were sacrificed at 7 weeksof age.

The results are summarized in FIG. 14; Y-axis represents % of totalcirculating monocytes among circulating immune cells. The results showthat the percentage of blood monocytes was increased in 5XFAD-Ctrl whencompared with WT mice. The results also show that by administeringZLN005 to AD transgenic animals, the percentage of monocytes decreased.ANOVA was used for statistical analysis.

Example 15. Ppargc1a Activator ZLN005 Improves Fine Motor Skills in ADTransgenic Animals

As described in Example 7, nest-building skill is one of the mostreliable measurements of motor function. In this test, AD transgenicanimals were orally treated 3 times a week for 4 weeks with 0.5%methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose,starting at 3 weeks of age. At 7 weeks of age, animals were given cottonpads and the amount of cotton that was shredded over a 24-hour periodwas measured. The nest building activities (g) are shown in FIG. 15.ZLN005 treatment (n=10) significantly increased the amount of cottonshredded by 5XFAD-ZLN animals in comparison to 5XFAD-Ctrl animals (n=8),indicating that motor skills of AD animals were improved. Unpairedt-test was used for statistical analysis.

C. Examples 16-21 Relate to ALS

WT=wild-type animals, ALS-Ctrl=transgenic ALS animals treated with 0.5%methylcellulose, ALS-ZLN=transgenic ALS animals treated with ZLN005.

Example 16. Ppargc1a Activator ZLN005 Suppresses Neuroinflammation inBrain Perivascular Macrophages in ALS Transgenic Animals

ALS transgenic animals were purchased from Jackson Laboratories. Theseanimals express the G93A mutation in the gene SOD1 which has beenimplicated as the cause of the disease in a subset of human subjectswith familial ALS. The animals exhibit hind limb paralysis, a classicalsymptom of ALS, upon 100-110 days of age and rapidly succumb. Theseanimals represent a gold standard model for therapeutic discovery in thefield of ALS research.

ALS transgenic animals were orally treated 3 times a week for 8 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 5 weeks of age.

For study of brain perivascular macrophages, ALS-Ctrl (n=8), ALS-ZLN(n=12), and WT animals (n=12) were sacrificed at 13 weeks of age.PBS-perfused brain tissues of sacrificed animals were digested withCollagenase IV and processed for flow cytometry. Brain perivascularmacrophages were phenotyped with antibodies directed against mouse iNOS,IL6, and TNFα (Biolegend) for flow cytometric acquisition (LSRII, BD)and analysis (FlowJo).

The results are summarized in FIG. 16; Y-axis represents % of brainperivascular macrophages that express iNOS (A), IL6 (B) and TNFα (C).The results show that brain perivascular macrophages in ALS transgenicmice exhibit an inflammatory phenotype, evidenced by a significantincrease in iNOS, IL6, and TNFα production in ALS-Ctrl mice whencompared with WT animals. The results also show that by administeringZLN005 to ALS transgenic animals, iNOS production in the brainperivascular macrophages of these treated animals decreased and thusneuroinflammation was suppressed. IL6 and TNFα production in the brainperivascular macrophages of ZLN005 treated animals were also suppressed,although these differences did not reach statistical significance. ANOVAwas used for statistical analyses.

Example 17. ZLN005 Improves Motor Skills in ALS Transgenic Animals

ALS transgenic mice were orally treated 3 times a week for 4 weeks with0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 9 weeks of age.

A wheel-running test was performed similarly to that described inExample 8. The animals started training at 13 weeks of age for 1.5 weeksof training on a treadmill at a constant speed of 10 rpm and then for1.5 weeks of training at an accelerating speed from 5-15 rpm. After thetraining period at 14.5 and 16 weeks of age, animals were tested formotor performance at a constant speed and at an accelerating speed,respectively. The results are shown in FIGS. 17A-17B. The results showthat ALS-ZLN mice (n=16) exhibited significantly increased latency tofall than ALS-Ctrl mice (n=16) both at a constant speed (296.3 secondsvs. 261.4 seconds, FIG. 17A) and at an accelerating speed (243.7 secondsvs. 118.1 seconds, FIG. 17B). Unpaired t-tests were used for statisticalanalyses.

Example 18. ZLN005 Improves Survival Rate in ALS Transgenic Animals

ALS transgenic mice were orally treated 3 times a week with 0.5%methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose,starting at 5, 10 and 15 weeks of age.

Survival of ALS-Ctrl (n=18) and ALS-ZLN mice (n=8) were monitored untilall animals succumbed. The results (FIG. 18) demonstrate that ALS-ZLN at5 or 10 weeks of age significantly increased survival (mean survival of131-132 days) in comparison to ALS-Ctrl (mean survival of 119 days);p-values<0.05. Log-rank test was used for statistical analysis.

Example 19. Ppargc1a Activator ZLN005 Suppresses Perivascular MacrophageAccumulation in the Brains of ALS Transgenic Animals

ALS transgenic animals were orally treated 3 times a week for 8 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 5 weeks of age.

For measuring the frequency of CD45hi CD11b+F4/80+brain perivascularmacrophages, ALS-Ctrl (n=13), ALS-ZLN (n=17), and WT animals (n=17) weresacrificed at 13 weeks of age. PBS-perfused brain tissues of sacrificedanimals were digested with Collagenase IV and processed for flowcytometry.

The results are summarized in FIG. 19; Y-axis represents % of brainperivascular macrophages among total brain immune cells in the brain.The results show an increase in the percentage of brain perivascularmacrophages in ALS-Ctrl mice when compared with WT mice. The resultsalso show that by administering ZLN005 to ALS transgenic animals, thepercentage of the brain perivascular macrophages of these treatedanimals decreased. ANOVA was used for statistical analysis.

Example 20. Ppargc1a Activator ZLN005 Suppresses Glycolytic Activationin Brain Perivascular Macrophages in ALS Transgenic Animals

ALS transgenic animals were orally treated 3 times a week for 8 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 5 weeks of age.

For measuring brain perivascular macrophages, ALS-Ctrl (n=8), ALS-ZLN(n=8), and WT animal (n=12) were sacrificed at 13 weeks of age.PBS-perfused brain tissues of sacrificed animals were digested withCollagenase IV and processed for flow cytometry. Brain perivascularmacrophages were stained with 2-NBDG, the fluorescent glucose analog, tomeasure glucose uptake for flow cytometric acquisition (LSRII, BD) andanalysis (FlowJo).

The results are summarized in FIG. 20; Y-axis represents % of brainperivascular macrophages that have taken up the glucose analog, 2-NBDG.The results show that brain perivascular macrophages in ALS transgenicmice exhibited a glycolytic phenotype, evidenced by a significantincrease in 2-NBDG uptake in ALS-Ctrl mice when compared with WT mice.The results also show that by administering ZLN005 to ALS transgenicanimals, glucose uptake in the brain perivascular macrophages of theseALS-ZLN animals decreased and thus glycolytic activation and metabolicdysfunction in brain perivascular macrophages in ALS transgenic animalswere suppressed. ANOVA was used for statistical analysis.

Example 21. Ppargc1a Activator ZLN005 Suppresses Systemic Inflammationin ALS Transgenic Animals

ALS transgenic animals were orally treated 3 times a week for 8 weekswith 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5%methylcellulose, starting at 5 weeks of age.

For measuring the frequency of CD115+CD11b+ blood monocytes, ALS-Ctrltransgenic animals (n=10), ALS-ZLN (n=10), and WT animal (n=10) weresacrificed at 13 weeks of age.

The Y-axis in FIGS. 21A and 21B represents % of total monocytes and % ofLy6C+ inflammatory monocytes among circulating immune cells. The resultsshow that monocytes, especially the Ly6C+ subset, were increased inALS-Ctrl n compared with wild-type mice. The results also show that byadministering ZLN005 to ALS transgenic animals, the percentage of thesecells in treated animals decreased and thus systemic inflammation wassuppressed. FIG. 21C shows that serum levels of TNF-α measured by ELISAin ALS transgenic animals were significantly suppressed by ZLN005treatment Unpaired t-tests and ANOVA were used for statistical analyses.

D. Example 22 relates to HD.

Ctrl=transgenic HD animals treated with 0.5% methylcellulose,ZLN=transgenic HD animals treated with ZLN005.

Example 22. ZLN005 Improves Motor Skills in HD Transgenic Animals

The wheel-running test in this example was performed similarly to thatdescribed in Example 8.

HD transgenic animals (R6/2) were purchased from Jackson Laboratories.The animals exhibit symptoms of HD such as hind limb paralysis, musclewasting, and impaired motor coordination, upon 8-10 weeks of age andrapidly succumb. HD transgenic mice were orally treated 3 times a weekfor 4 weeks with either 0.5% methylcellulose (n=8) or ZLN005 (Sigma) at25 mg/kg in 0.5% methylcellulose (n=7), starting at 6 weeks of age.Subsequently, at 10 weeks of age, these mice were subjected to 2 weeksof training on a treadmill at a constant speed of 5 rpm (rotations perminute). After the training, mice were tested for motor performance at aconstant speed of 5 rpm. The results are shown in FIG. 22. The resultsshow that HD-ZLN005 animals had a significantly increased latency tofall when compared with HD-Ctrl mice (110 seconds vs. 41.7 seconds).Unpaired t-test was used for statistical analysis.

E. Examples 23-24 Relates to DLB

Ctrl=DLB transgenic animals treated with 0.5% methylcellulose, ZLN=DLBtransgenic animals treated with ZLN005. FF=Ppargc1a^(LoxP/LoxP) mice onDLB transgenic background, Cre=Ppargc1a^(LoxP/LoxP)Cx3cr1^(CreER) miceon DLB transgenic background.

Example 23, Microglia-Specific Deletion of Ppargc1a Worsens MotorDysfunction in Transgenic DLB Animals

SNCA.*A53T transgenic mice, an animal model in which the mutated form ofhuman alpha synuclein is overexpressed, were generated to studypathological mechanisms in PD and DLB (Lee et al, Proc Natl Acad SciUSA. 2002, 13:8968-8970), These animals exhibit accumulation ofpathogenic Lewy bodies upon aging, resulting in progressive motordysfunction and eventual death.

Animals with microglia-specific deletion of Ppargc1a were generated asdescribed in Example 1. Furthermore, these animals were bred withSNCA*A53T animals to generate mice with microglia-specific deletion ofPpargc1a on DLB genetic background. After tamoxifen treatment to inducedeletion of Ppargc1a in microglia, animals were rested for 5 weeksbefore being subjected to treadmill training. At 8 weeks of age, theseanimals were subjected to 1.5 weeks of training on a treadmill at aconstant speed 10 rpm (rotations per minute) and then 1.5 weeks oftraining at an accelerating speed from 5-15 rpm as described in Example8. After the training period at 9.5 and 11 weeks of age, animals weretested for motor performance at a constant speed and at an acceleratingspeed, respectively.

The results, shown in FIG. 23A-23B, are representative of twoindependent experiments of one animal per genotype with similaroutcomes. FF animals exhibited significantly longer latency to falls(average of two running trials) than Cre animals, which had Cre-mediateddeletion of Ppargc1a in Cx3cr1 expressing microglia, at both constantspeed of 10 rpm and accelerating speed of 5-15 rpm. These results showthat microglia-specific Ppargc1a protects against motor dysfunction inthis transgenic model of DLB.

Example 24, Ppargc1a Activator ZLN005 Improves Motor Skills in DLBTransgenic Animals

DLB transgenic animals were purchased from Jackson Laboratories and wereorally treated 3 times a week with 0.5% methylcellulose (Ctrl) or ZLN005(ZLN) at 25 mg/kg in vehicle, starting at 8 weeks of age for 12 weeks.

Subsequently, at 20 weeks of age, these animals were subjected to 1.5weeks of training on a treadmill at a constant speed 10 rpm and then 1.5weeks of training at an accelerating speed from 5-15 rpm, similar tothose described in Example 8. After the training period at 21.5 and 23weeks of age, animals were tested for motor performance at a constantspeed and at an accelerating speed, respectively.

The results are shown in FIG. 24A-24B. DLB-ZLN mice (n=11) show anincrease in latency to fall in comparison to DLB-Ctrl mice (n=9) at aconstant speed of 10 rpm (271.5 seconds vs. 252.8 seconds). However,this difference did not reach statistical significance. At theaccelerating speed of 5-15 rpm, DLB-ZLN mice performed significantlybetter than DLB-Ctrl mice (194.0 seconds vs. 134.5 seconds, pvalue<0.05). Thus, motor dysfunction of DLB mice was alleviated by N005treatment. Unpaired t-tests were used for statistical analyses.

Example 25, Ppargel a Activator ZLNOO5 Improves Motor Skills andSurvival in FTI) Transgenic Animals (Prophetic Example)

TARDBP*A315T transgenic mice have been generated as an animal model tostudy ALS and FTD. These animals overexpress a mutant form of the DNAbinding protein TARDBP, whose cytoplasmic inclusions are present in thebrains of subjects with ALS and FTD (Barmada et al Nat Chem. Biol.,10:677-685, 2014). Ke et al (Short-term Suppression of A315T MutantHuman TDP-43 Expression Improves Functional Deficits in a NovelInducible Transgenic Mouse Model of FTLD-TDP and ALS, Acta Neuropathol.2015 Oct 5, e-Publication) report that constitutive expression ofTARDBP*A315T resulted in progressive neurodegeneration, and compromisedmotor performance, spatial memory and disinhibition. This model has beenwidely used for screening of compounds with therapeutic potentials inALS and FTD.

These FTD transgenic animals are purchased from Jackson Laboratories andare orally treated 3 times a week with 0.5% methylcellulose (FTD-Ctrl)or ZLN005 (FTD-ZLN) at 25 mg/kg in vehicle, starting at 6 weeks of age.Subsequently, at 10 weeks of age, these animals are subjected to 1.5weeks of training on a treadmill at a constant speed 10 rpm and then 1.5weeks of training at an accelerating speed from 5-15 rpm. After thetraining period at 11.5 and 13 weeks of age, animals are tested formotor performance at a constant speed and at an accelerating speed,respectively. Finally, they are monitored for survival analysis.

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims.

1.-12. (canceled)
 13. A method of suppressing microglia-mediated inflammation in a subject diagnosed with a neurodegenerative disease, comprising the step of administering to a subject suffering from a neurodegenerative disease an effective amount of a solvate or pharmaceutically acceptable salt of 2-(4-tert-butylphenyl)-1H-benzimidazole.
 14. The method of claim 13, wherein the neurodegenerative disease is selected from the group consisting of: amyotrophic lateral sclerosis (ALS), Alzheimer disease, Parkinson's disease, Huntington's disease, frontotemporal degeneration, dementia with Lewy bodies, a motor neuron disease, and a demyelinating disease.
 15. The method according to claim 13, wherein said compound is administered by systemic administration.
 16. The method according to claim 13, wherein said compound is administered by oral administration.
 17. The method of claim 14, wherein the neurodegenerative disease is ALS.
 18. The method of claim 14, wherein the neurodegenerative disease is Alzheimer's disease.
 15. The method of claim 14, wherein the neurodegenerative disease is Parkinson's disease.
 19. The method of claim 14, wherein the neurodegenerative disease is Huntington's disease.
 20. The method of claim 14, wherein the neurodegenerative disease is frontotemporal degeneration.
 21. The method of claim 14, wherein the neurodegenerative disease is dementia with Lewy bodies.
 22. The method of claim 14, wherein the neurodegenerative disease is a motor neuron disease.
 23. The method of claim 14, wherein the neurodegenerative disease is a demyelinating disease.
 24. A method of treating dementia with Lewy bodies, comprising the step of administering to a subject suffering from dementia with Lewy bodies an effective amount of 2-(4-tert-butylphenyl)-1H-benzimidazole, or a solvate or pharmaceutically acceptable salt thereof.
 25. The method according to claim 24, wherein the method alleviates motor dysfunction and/or cognitive decline in the subject. 